Thyroid cancer is increasingly prevalent and the most common malignancy of endocrine tissues, and most patients require lifelong surveillance with diagnostic thyroid-stimulating hormone (TSH)-stimulated radioiodine uptake as well as thyroid remnant or tumor ablation with radioiodine therapy. The PI, who was both co-inventor and co-developer with Genzyme of wild type recombinant human (rh) TSH (Thyrogen) currently approved for stimulation of diagnostic radioiodine uptake and thyroglobulin secretion, now proposes to develop a more potent and more efficacious second generation superactive analog of rh TSH for the 15-20% of poorly responsive thyroid cancer patients in diagnostic imaging as well as for the majority of such patients for radioiodine therapy. Our previous structure-function studies resulted in the discovery of the first analogs of TSH and other glycoprotein hormones with major increases in receptor binding affinity and bioactivity. During our highly scored and successfully completed phase 1 SBIR study we have achieved all the aims including developing stable cell lines producing high levels of the final two candidate superactive analogs; optimization of large scale bioreactor production methods; development of a novel, high capacity purification methods suitable for commercial scale-up; rigorous quantification and characterization of purified analogs by multiple physicochemical methods, and proof of increased potency and efficacy in the now classic in vivo mouse rh TSH in vivo bioassay first developed by the PI. In the current phase 2 proposal, for which we have provided extensive preliminary data well beyond the aims of the phase 1 proposal, we plan to produce and purify additional large amounts (>200mg) of the final two TSH analog candidates TR1401 and 1402 in the fibrous bed bioreactor sufficient for all the phase two in vivo studies using the methods of production and purification optimized in the phase one study. We also plan to develop and validate a novel immunoassay for the detection of TSH analogs in unpurified rodent plasma that quantifies accurately and identically to rigorous HPLC methods which will then be used to determine the IV metabolic clearance rate and the IP, SC and IM phamacokinetics in rodents. Most importantly, in this phase two proposal we will examine for the first time multiple clinically relevant diagnostic and therapeutic endpoints of TSH action comparing TSH analogs TR1401 and 1402 to wild type (Genzyme) recombinant TSH in normal mice and in those with Ret/PTC1 and other transgenic mouse models of thyroid cancer. Specifically we will examine detailed dose response curves for both wild type and analog TSH in thyroidal 125I uptake by direct organ counting, diagnostic thyroidal PET imaging with 124I and 18 Fluorodeoxyglucose as well as therapeutic 131I -induced tumor regression. Finally, we will determine the possible complementation of the diagnostic and therapeutic effects of TR1401 and 1402 with those of selected newly developed protein kinase inhibitors in vitro and in vivo. PUBLIC HEALTH RELEVANCE: Thyroid cancer is the most common malignancy of endocrine tissues and most patients require lifelong surveillance with diagnostic thyroid-stimulating hormone (TSH)-stimulated radio-iodine imaging to detect recurrent tumor. The PI and Co- Investigator have developed a much improved form of TSH and propose a completely new method of thyroid cancer imaging that should greatly improve early detection and lead to improved cancer treatment and survival. [unreadable] [unreadable] [unreadable]